飞蝗几丁质酶基因家族的功能及激素调控机制研究

Chitin is widely distributed in epidermis and the peritrophic matrix of insects. The importance of chitin in insect development and the absence of chitin in vertebrates have led to a hypothesis of attacking insect chitin biosynthesis pathway as a novel target for developing safe and effective insecticides...Insect chitinases (EC 3.2.1.14, endochitinases) are enzymes catalyzing random hydrolysis of N-acetyl-?-D-glucosamine in chitin and chitodextrins during insect molting process. Multiple chitinase genes have been identified in various insect species, however, the gene numbers and their roles in insect development have not been well elucidated, which strongly limited the application of insect chitinase genes in pest control. ..In our project, chitinase genes of Locusta migratoria will be studied, the locust EST and genome database provide a good platform to conduct this research work. The partial cDNA sequences of chitinase genes could be obtained from Locust transcriptom and genome database by using bioinformatic methods; The full-length cDNA sequences were cloned by RACE-PCR technology, the phylogenetic tree was constructed by comparing with other known insect chitinase amino acid sequences, after that, we can carry out nomenclature of Locust chitinase genes according to properties of sequences and phylogenetic analysis; Tissue and developmental expressions will be determined by using RT-PCR and Real-time PCR; In situ hybridization and immunohistochememistry were used for localization of chitinase gene expression; The RNA interference will be performed to explore the biological function of chitinase genes; We further found that the developmental expression patterns of chitinase gene were correlated well with the change of 20-hydroxyecdysone (20E) titers from the 1-day forth-instar nymphs to the 7-day adults, the mechanism of hormone regulation will be further investigated in our study. These results will provide strong scientific data for developing novel strategies for controlling this important pest, the enzymes encoded by these genes could potentially serve as an excellent target for pest control.

几丁质酶是昆虫蜕皮过程中的关键酶，人类和高等动物不存在几丁质，因此该酶是昆虫特有的，作为害虫防治的靶标相对安全。昆虫几丁质酶基因的数量及功能分化问题尚未得到解决，使其在应用中受到很大制约。本项目拟采用生物信息学方法，利用飞蝗转录组和基因组的数据平台，获得飞蝗几丁质酶基因部分片段；通过RACE-PCR等技术获得基因cDNA全长，与已知昆虫几丁质酶基因构建系统发育树，获得飞蝗几丁质酶基因的系统分类和科学命名；应用RT-PCR和Real-time PCR技术分析飞蝗丁质酶基因的表达特性；采用原位杂交和免疫组化对几丁质酶基因的表达进行细胞定位；通过注射和饲喂等不同方法开展系统的RNAi研究，阐明飞蝗几丁质酶基因的功能特性；进一步分析飞蝗发育过程中激素滴度和几丁质酶基因表达的相关性，深入探讨飞蝗不同几丁质酶基因的激素调控机制。研究结果将对基于几丁质代谢途径和激素调控研发害虫防治的新方法提供理论依据。

几丁质是昆虫表皮重要的组成成分，对蜕皮发育起关键作用。因人类和其他高等动物体内没有几丁质，针对其代谢途径进行害虫防治相对安全。几丁质酶（Chitinase, EC3.2.1.14）是昆虫几丁质降解过程的关键酶，由多基因编码，广泛参与发育、消化和免疫等生命活动。然而昆虫几丁质酶基因的数量及功能尚不明确。本研究以重要农业害虫飞蝗（Locusta migratoria）为对象，通过RACE-PCR获得16条几丁质酶基因cDNA全长；构建系统发育树并科学命名；不同组织部位表达图谱显示：LmCht5-1、LmCht5-2和LmCht10在体壁、前肠和后肠中表达较高，可能参与表皮几丁质的降解；卵期发育天数的表达图谱暗示3个基因可能在不同发育阶段起作用。飞蝗具有2条Ⅰ组基因（LmCht5-1与LmCht5-2），LmCht5-1具有几丁质结合域，而LmCht5-2缺失这一结构域。聚类分析表明二者与其他昆虫Cht5直系同源，且关键位点的氨基酸残基“DWEYP”未发生突变；真核表达发现两个蛋白均具有酶活性，LmCht5-1与胶体几丁质结合能力更强。RNAi显示两个基因功能出现分化：LmCht5-1缺失可影响5龄若虫正常蜕皮，而LmCht5-2缺失无影响。RNAi沉默LmCht10后，任一龄期飞蝗若虫均出现蜕皮困难致死表型；几丁质酶活性测定、体壁组织切片及几丁质含量测定等表明该基因主要降解旧表皮中的几丁质，是蜕皮过程的必需基因。成虫LmCht10沉默后，产卵量下降，且若虫孵化后卵包膜无法蜕去而致死。5龄若虫发育过程中蜕皮激素滴度发生变化，与LmCht5-1、LmCht5-2和LmCht10的发育表达图谱趋势一致。沉默蜕皮激素受体和注射20-羟基蜕皮酮（20-hydroxyecdysone，20E）的实验结果表明3个基因的表达均严格受蜕皮激素调控。筛选获得mi-263负调控LmCht10，系统解析了其分子调控机制。本项目筛选获得高效抗虫的几丁质酶分子靶标LmCht5-1和LmCht10，并深入解析其激素及miRNA的分子调控机制。研究成果将有力促进抗虫分子靶标创新及植保新技术的研发，具有重要的理论意义和实践应用价值。